Abstract

Our
objective was to determine whether an immune response can be generated
against MUC1 peptide and against tumor cell MUC1 after vaccination with
MUC1-keyhole limpet hemocyanin (KLH) conjugate plus QS-21 in breast
cancer patients.

Nine patients with a history of breast cancer but without evidence of
disease were treated with MUC1-KLH conjugate plus QS-21, containing 100μ
g of MUC1 and 100 μg of QS-21. s.c. vaccinations were administered
at weeks 1, 2, 3, 7, and 19. Peripheral blood was drawn at frequent
intervals to assess antibody titers. Skin tests were placed at weeks 1,
3, 9, and 21 to determine delayed type hypersensitivity reactions.

Common toxicities included a local skin reaction at the site of the
vaccine, usually of 4–5 days’ duration, and mild flu-like symptoms
usually of 1–2 days’ duration. High IgM and IgG antibody titers
against synthetic MUC1 were detected. IgG antibody titers remain
elevated from a minimum of 106–137 weeks after the first vaccination.
Binding of IgM antibody to MCF-7 tumor cells was observed in seven
patients, although there was minimal binding of IgG antibody. Two
patients developed significant antibody titers post-high-dose
chemotherapy and stem cell reinfusion. There was no evidence of T cell
activation.

This MUC1-KLH conjugate plus QS-21 was immunogenic and well tolerated
in breast cancer patients. Additional trials are ongoing to determine
the optimal MUC1 peptide for use in larger clinical trials. Further
investigation of vaccine therapy in high-risk breast cancer is
warranted.

INTRODUCTION

Despite recent advances, current therapeutic regimens for both
metastatic and early stage breast cancer remain suboptimal. Standard
therapy for metastatic breast cancer results in minimal impact on
survival (1, 2)
, whereas adjuvant therapy reduces the
annual risk of recurrence by approximately one-third (3, 4)
. Patients at a particularly high risk for relapse include
those with a history of metastatic (5, 6, 7)
or locally
advanced breast cancer (8)
and those with increasing
levels of tumor markers, including
CEA,3
CA15-3 or BR2729
(9, 10, 11)
. In an attempt to provide greater clinical
benefit, evaluation of an immunotherapeutic approach has generated
considerable enthusiasm (12)
.

Investigation of vaccines for potential therapeutic purposes has been
accelerated by technological advances and the identification of various
differentiation antigens found on breast cancer cells. For example,
carbohydrates (i.e., gangliosides and blood-group related
antigens), and peptides are possible antigenic targets
(13, 14, 15, 16)
. Of particular interest is the mucin MUC1, which
is a transmembrane glycoprotein composed of a polypeptide core
containing multiple tandem repeats of a 20-amino acid sequence with
numerous carbohydrate side chains (17)
. MUC1 was initially
characterized by the development of murine monoclonal antibodies
reactive with mucins from human milk or malignant cells derived from
the breast or pancreas (17, 18)
. It is commonly found on a
variety of normal epithelial cells, including lung, breast, pancreas,
stomach, colon, salivary gland, kidney, endometrium, and prostate
(16, 18, 19)
, as well as malignant cells of breast, ovary,
pancreas, endometrium, colon, lung, and prostate origin (16, 18, 19, 20)
.

However, there are differences between MUC1 expression on normal cells
and tumors. For example, the monoclonal antibody SM3 demonstrates
significant reactivity with MUC1 on paraffin embedded breast cancer but
minimal reactivity with normal breast cells or benign breast lesions
(18, 21)
. On normal cells, mucins are located on the
apical surface and are extensively glycosylated. However, in tumors,
the usual structure of the tissue is disrupted so that mucin may be
found on multiple cell surfaces (22)
. In addition,
abnormal glycosylation in the cancer results in less complex and fewer
carbohydrate side chains (23)
. Therefore, in tumors, there
is greater exposure of MUC1 epitopes to the immune system, compared to
normal cells (24)
. Although the function of MUC1 is not
clearly established, it may allow for tumor growth through its
interactions with adhesion molecules and lymphocytes (22)
.
The cloning and sequencing of MUC1 led to the production of synthetic
peptides of various lengths for use in vaccines (25)
.

There are preclinical data in rodents demonstrating that MUC1 based
vaccines are immunogenic in both a humoral and cellular fashion. For
example, inhibition of tumor growth has been associated with antibody
production (26, 27, 28)
and T cell activation
(29, 30, 31)
. Vaccination of chimpanzees with MUC1 vaccine
constructs has also resulted in an immunogenic response (32, 33)
.

In addition, there is evidence that MUC1 is occasionally immunogenic in
un-vaccinated breast cancer patients. First, MHC unrestricted cytotoxic
T cells that lyse breast tumor cells but not normal breast cells have
been derived from axillary lymph nodes of patients with breast cancer
(34)
. Second, a humoral response directed against MUC1 has
also been noted. In patient sera, IgM antibodies reactive with
synthetic MUC1 peptides and circulating immune complexes containing
MUC1 have been detected (35, 36)
. Despite these findings,
the development of an immune response against MUC1 on tumor cells may
be limited because of low tumor immunogenicity.

An effective method of inducing antibodies against weak immunogens is
conjugation with a protein carrier, such as KLH, and the use of an
immune adjuvant, such as QS-21. KLH is a large immunogenic protein
obtained from the blood of the keyhole limpet. QS-21 is a saponin
fraction derived from the bark of the South American tree
Quillaja saponaria Molina (37)
.
This combination of KLH and QS-21 is an effective regimen for inducing
antibody responses against a variety of antigens.

For example, in preclinical analyses, high antibody titers
reactive with MUC1 peptide and tumor cells expressing MUC1 were
observed after vaccination of mice with synthetic MUC1 peptides
conjugated to KLH and mixed with QS-21 (28)
. In addition,
inhibition of tumor growth was noted in these mice after injection with
E4 cells (MUC1-expressing mammary cell line), despite the lack of a
detectable T cell response. In contrast, vaccination with unconjugated
MUC1 peptides mixed with either BCG or QS-21 yielded minimal antibody
responses. In clinical trials, vaccination of melanoma patients with a
variety of ganglioside KLH conjugates plus QS-21 has also yielded
higher antibody titers than vaccines containing the same gangliosides
with other immune carriers or adjuvants (38)
. A dose of
100 μg of QS-21 has been established as effective and well tolerated
(39)
.

Based on these studies, we initiated a trial in high-risk breast
cancer patients with no clinical evidence of disease. The primary
objective was to determine whether an immune response could be elicited
against MUC1 peptide and against tumor cell MUC1 after vaccination with
the MUC1-KLH conjugate plus QS-21. Based on statistical models, a
relatively small number of patients were enrolled to answer this
question (40)
.

PATIENTS AND METHODS

Breast cancer patients without evidence of disease and with one of
the following characteristics were potentially eligible for the study:
(a) AJCC stage IV disease after eradication of all
detectable disease by surgery, radiation, or chemotherapy;
(b) AJCC stage I, II, or III disease with rising tumor
markers (CA15-3, BR2729, or CEA); or (c) AJCC stage III
disease with an initially unresectable primary tumor after completion
of adjuvant therapy within 12 months. A rising CA15-3 or CEA level was
defined as a previously normal value with a subsequent elevation above
the normal range documented on two consecutive occasions at least two
weeks apart. For patients with a significant smoking history and a
chronically elevated CEA level < 15 ng/ml, a rising CEA level was
defined as one that increased at least 1.5× the uppermost chronic
value on two consecutive occasions at least 2 weeks apart.

Chemotherapy, radiation therapy, or surgery must have been completed at
least 4 weeks prior to treatment and immunotherapy within 6 weeks.
Hormonal therapy was allowed during the study. Other requirements
included: Karnofsky performance status >80%, lymphocyte count≥
0.5 × 106/ml, no other active cancers,
and no history of a seafood allergy. Premenopausal patients were
required to have a normal β-human chorionic gonadotropin level.

A history and physical examination, rectal examination with Hemoccult,
chest X-ray, complete blood count with differential, CEA, CA15-3 (or
BR2729), creatinine, chemistry profile, and gamma
glutamyltranspeptidase level were required within three weeks of
treatment. A CT scan of the chest/abdomen/pelvis and bone scan were
obtained within four weeks of treatment. A colonoscopy within at least
5 years was required for patients with an isolated elevation of the CEA
level. Patients were required to sign an informed consent form that had
been approved by the Institutional Review Board and the Food and Drug
Administration.

Treatment Plan and On-study Evaluation.

All patients received MUC1-KLH conjugate plus QS-21, containing 100μ
g of MUC1 and 100 μg of QS-21 via s.c. vaccination. A total of
five s.c. vaccinations were administered at weeks 1, 2, 3, 7, and 19 as
an outpatient in the Immunology Unit at rotating sites, including the
arm, thigh, or buttocks (Table 1)⇓
.

A history and physical examination, complete blood count with
differential, chemistry profile, CEA, and CA15-3 (or BR2729) levels
were obtained intermittently as outlined in Table 1⇓
. A bone scan and CT
scans of the chest/abdomen/pelvis, CEA, and CA15-3 (or BR2729) levels
were obtained at weeks 21–24 for patients with elevated tumor markers
and for other patients at the discretion of their primary physician.
All patients were asked to complete a diary after each vaccination to
document any symptoms.

Peripheral blood to assess immune response was drawn at week 1
(pretherapy) and at time intervals as noted in Table 1⇓
. Sera was
analyzed by ELISAs for antibody titers against synthetic MUC1 and
against a cell line expressing MUC1 (MCF-7). Additional blood was drawn
at weeks 1, 5, 9, and 21 to assess for T cell response. Skin tests with
topical DNCB were placed at weeks 1, 3, 9 and 21 to assess for delayed
type hypersensitivity reactions. The DNCB dose was 2000 μg at the
first application and 100:50:25 μg for the second skin test, and
doses were decreased for subsequent applications based on
patient reaction. The protocol was later amended to also include
intradermal skin tests with 30 μg of MUC1 at weeks 1, 3, 9, and 21.
The area of skin discoloration at the site of the skin test was
measured at 48 h by either the patient or the nurse. Patients were
followed every 3 months if feasible.

Vaccine Preparation.

The primary antigenic component of this vaccine contains 1.5 repeats of
the 20-amino acid sequence of the MUC1 peptide. This 30-amino acid
sequence is C-VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA. Attached to the
N-terminal carboxyl end is cysteine (Fig. 1)⇓
. This MUC1 peptide was synthesized in
an automated peptide synthesizer at the Memorial Sloan-Kettering Cancer
Center. The other vaccine components were obtained from the following
sites: MBS from Pierce (Rockford, IL); KLH from PerImmune Inc.
(Rockville, MD); and QS-21 from Aquilla Biopharmaceutical, Inc.
(Worcester, MA).

Components of MUC1-KLH conjugate plus QS-21. C,
cysteine. The 30-amino acid sequence for the MUC1 peptide is noted in
the inner box.

Production of the vaccine began with a mixture of MBS/dimethylformamide
and KLH as described previously (28)
. The MBS is a
bifunctional linker that facilitates covalent binding of the KLH to the
terminal cysteine on the MUC1 peptide. This mixture was passed through
a Sephadex G25 column to remove unconjugated MBS. The remaining MBS-KLH
was then combined with MUC1 peptide and incubated for 3 h. A
molecular weight 30,000 Centriprep filter was then used to
eliminate free MUC1 peptide (Amicon Inc., Beverly, MA). The conjugate
MUC1:KLH epitope ratio was 560:1. The MUC1-KLH conjugate was mixed with
QS-21, placed in a vial, and tested for sterility, purity,
immunogenicity, and safety.

Dose Modifications and Off-study Criteria.

Toxicity was graded according to the Common Toxicity Criteria. A 50%
reduction in doses of all vaccine components was planned for individual
patients with grade III or greater local or systemic toxicity.
Discontinuation of treatment was planned for recurrence of disease
requiring systemic therapy or radiation.

Serological Analysis.

ELISAs were performed to detect IgG and IgM antibody production against
MUC1. ELISA plates were coated with the MUC1 peptide (30- or 32-amino
acid sequence) at 0.1 μg/well, in carbonate buffer, and incubated at
4°C. overnight. To block unreacted sites, 3% human serum albumin was
added for 2 h. Serial dilutions of patient sera were then added to
the ELISA plates. After a 2-h incubation, the plates were washed, and
alkaline phosphatase labeled goat anti-human IgM antibody (Kierkegaard
and Perry Labs, Gaithersburg, MD) was added. For IgG detection,
unlabeled mouse anti-human IgG (Southern Biotechnology, Birmingham, AL)
was used instead of IgM antibody, followed by alkaline-phosphatase
labeled goat anti-mouse IgG (Southern Biotechnology). After a 45-min
incubation, the plates were washed and developed. The change in color
was measured at 414 nm on the ELISA reader. The antibody titer was
defined as the highest serum dilution with an absorbance of ≥0.100
(28)
. Measurement of IgG subclasses was determined by
ELISA assays using goat anti-human IgG1, IgG2, IgG3, or IgG4
labeled with alkaline phosphatase (Zymed, San Francisco, CA).

Flow cytometric assays were performed to determine whether the IgG or
IgM antibodies were binding to tumor cells. Tumor cells (1 ×
107) from the MCF-7 human mammary carcinoma cell
line (provided by Dr. Neil Rosen, Memorial Sloan-Kettering Cancer
Center), which expresses MUC1, or from the SKMEL 28 melanoma cell line,
which does not, were incubated with individual patient sera (1:5
dilution) from pretreatment samples (in all patients except week 2 for
patients 2 and 4) and from posttreatment samples with elevated antibody
titers. After washing, 20 μl of 1:25 dilution of FITC-labeled goat
anti-human IgM or IgG antibody (Southern Biotechnology) was
added, as described previously (28)
. After a 30-min
incubation on ice, the percentage of positive cells was determined by
flow cytometry (EPICS Profile II, Coulter Co., Hialeah, FL). The
monoclonal antibody HMFG-2, generously provided by Dr. Joyce Taylor
Papadimitrou, was the positive control against MUC1.

Assays for T-lymphocyte Immunity.

Cell mediated immunity was determined using limiting dilution chromium
release assays performed in the laboratory of Dr. Olivera Finn
(University of Pittsburgh School of Medicine, Pittsburgh, PA) with
cryopreserved peripheral blood mononucleocytes obtained pre- and
postvaccination for the initial six patients treated. After a 10-day
in vitro cultivation period with the MUC1 positive cell line
CAMA-1, MHC unrestricted cytotoxicity against a second MUC1 positive
but MHC distinct cancer cell line (BT-20) was tested as described
previously (32)
. K562 cells were used to block nonspecific
cytotoxicity.

RESULTS

Between June 1995 and June 1996, nine patients were enrolled in
this study. All patients had a history of breast cancer that was
histologically confirmed yet had NED at the time of protocol entry. The
patient characteristics are noted in Table 2⇓
. Eight patients had metastatic disease
documented previously (one in lung, four on chest wall, and three in
supraclavicular lymph nodes), and one patient had a mild elevation in
the CEA level of 6–11 ng/ml (normal range, 0–5.0 ng/ml). Three
patients had metastatic disease at the time of initial diagnosis. Most
patients had received prior chemotherapy, and two (patients 3 and 8)
had been treated with high-dose chemotherapy and stem cell reinfusion
for metastatic disease. All patients received hormonal therapy during
this trial, except for patient 5, who was Stage 4 NED. All patients
continue to be followed intermittently.

Toxicities.

Common toxicities are outlined in Table 3⇓
. All patients received five
vaccinations each, at the full dose. The most common toxicities were
local skin reactions and mild flu-like symptoms. Erythema, discomfort,
swelling, and pruritus were noted at the vaccination site in most
patients ranging from approximately 2 to 9 days in duration, although
these symptoms were frequently resolved within 4–5 days. The
discomfort was usually alleviated with acetaminophen. Two patients
developed blisters at the vaccine site (one patient after dose 2 and
one patient after dose 3), which resolved without scarring. At the
vaccine site, erythema measuring ≥15 cm was common. Mild flu-like
symptoms, including low-grade fever, headaches, chills, myalgias, and
fatigue, were self-limiting, usually of 1–2 days’ duration.
Approximately 3 days after the third vaccine, one patient reported
grade 2 nausea and grade 3 emesis, in association with a headache. This
was felt to be consistent with her history of migraine headaches, and
no dose reduction was made. Two patients noted a transient mild
increase in inguinal adenopathy near the vaccine site, and three
patients noted a skin recall reaction at earlier vaccine sites after
later vaccinations.

Common toxicities: number of episodes that
occurred following all vaccinations

Total number of vaccinations, 45.

No hepatic or renal toxicity was observed. No significant hematological
toxicity was noted during the study. Two patients developed transient
leukopenia (one with a pretreatment grade 1 value), and one patient
developed a transient grade 2 neutropenia (pretreatment grade 1 value).
The lowest hematological values throughout the entire study for each
patient are noted in Table 4⇓
.

There was no clinical evidence of an autoimmune reaction as determined
by symptoms, physical examination, or laboratory findings.

Immunological Response.

IgM and IgG antibody titers against MUC1 were evaluated by ELISA for
each patient at the time points mentioned previously. If feasible,
blood samples were also obtained approximately every 3 months after
completion of the study. The values of reciprocal titers are outlined
for each patient in Fig. 2⇓
. All patients
developed a significant increase in both IgM and IgG antibody titers
after vaccination. The IgG titers remain elevated in all patients at a
minimum of 106 weeks from the first vaccination. Analysis of IgG
subclass titers for anti-MUC1 antibodies were performed for each
patient using samples with high IgG titers in comparison to the
pretreatment value (data not shown). IgG1 and IgG3 were detected in all
patients postvaccination. Three patients had a minimal increase in
IgG2. IgG4 was not detected in any patient.

To determine whether these antibodies were able to bind to MUC1
naturally expressed on tumor cells, flow cytometric analysis was
performed to test IgM and IgG reactivity of patient sera against MCF-7
cells. Pre- and posttreatment values for all patients are noted in
Tables 5⇓
and 6⇓
. Seven patients (all but patients 2 and
4) demonstrated a clear increase in IgM reactivity against MCF-7 cells
after vaccination. However, there was minimally significant increase in
IgG reactivity against MCF-7 cells, with only three patients (patients
3, 4, and 9) demonstrating a tripling of the percentage of positive
cells.

BR2729 levels were obtained prevaccine therapy and during treatment.
These values were within the normal range for all patients at all of
these time points. Therefore, no clear correlation between antibody
titers and BR2729 levels could be made.

No delayed type hypersensitivity skin test reactivity against MUC1 was
detected. All patients had reactivity to the DNCB skin tests. There was
no evidence of increased precursor frequency of CTLs in the six
patients evaluated. Precursor frequencies ranged between 1 in 664,000
lymphocytes and 1 in 1,759,000 lymphocytes prior to vaccination, and
these values did not change significantly after vaccination.

Clinical Response.

Because all patients were without evidence of clinical disease at the
start of this study, tumor response was not an end point. The range of
follow-up from administration of the first vaccine was 106–162 weeks
(median, 135 weeks). However, two patients have developed a recurrence
during this time. Patient 5 underwent resection of a chest wall
recurrence in week 47 and was placed on hormonal therapy. She remains
without active disease. Patient 6 developed a chest wall recurrence in
week 80 and remains on hormone therapy with stable disease. All other
patients are without evidence of recurrence. The patient with the mild
CEA elevation continues to have a CEA level in the 6–7 ng/ml range.

DISCUSSION

Thus far, there has been minimal evaluation of vaccine
therapy in breast cancer patients. The majority of previous studies
noted a lack of significant clinical benefit after vaccine
administration with either tumor cells or general immune stimulants in
advanced disease (41, 42)
or in the adjuvant setting
(3, 43, 44)
. Overall, these unfavorable results were
likely caused by several factors, including small numbers of patients,
poorly defined eligibility criteria, and most importantly, limitations
of the agents used. However, recent clinical investigation of a sialyl
Tn glycoconjugate documented an immunogenic response with little
clinical toxicity (45)
. These results are encouraging and
support further exploration of vaccine therapy in breast cancer.

Several conclusions can be made from our study. First, vaccination of
breast cancer patients with the MUC1-KLH conjugate plus QS-21 was well
tolerated. The most common toxicities included transient local skin
reactions and mild flu-like symptoms. The changes in hematological
parameters are unlikely to be caused by the vaccine, as most patients
had low pretreatment values. The transient variation in values could be
attributable to normal biological fluctuations. Although mucin is
present on a variety of normal cells, we did not observe any evidence
of an autoimmune reaction. Several mechanisms may decrease the
potential for an autoimmune reaction or an endogenous anti-mucin
response. As noted previously, the glycosylation of mucins from normal
and malignant breast cells is different (34, 46, 47)
. In
addition, the secretion of mucin primarily on the luminal surface of
many normal epithelial cells limits its exposure to the immune system
(22, 34)
. These data support the suggestion that aberrant
glycosylation and cellular architecture may allow for greater exposure
of mucin epitopes to antibodies and T cells.

Second, vaccination of breast cancer patients with this vaccine results
in significant production of both IgM and IgG antibodies against
synthetic MUC1, but no evidence of T lymphocyte activation. For most
patients, the IgM and IgG antibody levels decreased with time. However,
IgG titers remain elevated from a minimum of 106–137 weeks after the
first vaccination. Whether these antibodies will eventually become
undetectable requires continued follow up of these patients. The
optimal number of doses and duration of vaccine administration has not
been established, and it is possible that patients may require
subsequent “booster” vaccinations.

Although many vaccine studies have focused on T cell activation to
elicit an antitumor response, there are significant data to indicate
that antibodies may also result in antitumor activity
(48)
. For example, increased disease-free and overall
survival associated with antibody production has been observed in
melanoma patients after vaccination with ganglioside vaccines
(49)
. It is too early to determine whether antibodies
against MUC1 or activation of T cells will correlate with improved
clinical outcome in breast cancer patients. It is not possible from
this small trial to conclude whether an association exists between
antibody titers and risk of relapse. Longer follow up with a larger
number of patients would be necessary to evaluate this point. As all
patients were without clinical evidence of disease, tumor was not
accessible for antibody detection.

Only a few clinical trials exploring various MUC1 based vaccines have
been conducted thus far. A Phase I trial at the University of
Pittsburgh treated breast cancer and gastrointestinal cancer patients
with a synthetic MUC1 peptide (105-amino acid sequence) plus BCG.
Toxicities were limited except for skin breakdown probably caused by
BCG. An increase in CTLs after vaccination was found in several
patients (50, 51)
. Another Phase I trial in Australia of a
mannan-MUC1 fusion protein in advanced cancer patients resulted in
minimal toxicity. High titers of anti-MUC1 IgG antibodies were found in
13 of 25 patients, and 4 of 15 patients had proliferation of T cells
(52)
. However, results of T cell assays can be difficult
to reproduce and clearly require further evaluation. It is unclear
whether the type of protein carrier (KLH) or immune adjuvant (QS21)
in our trial affected T cell activation.

Third, significant antibody titers were produced in both patient 3 and
patient 8, who had been treated with high-dose chemotherapy and stem
cell reinfusion approximately 44 and 48 months, respectively, prior to
the vaccine. Immunosuppression occurs after high-dose therapy, and it
may be prudent to allow a reasonable time period to elapse prior to
antitumor vaccinations. All other patients except patient 9 (no
prior chemotherapy) had completed chemotherapy 3–70 months prior to
the first vaccination. The median time from completion of chemotherapy
to the first vaccine for these eight patients was 45.5 months. Prior
chemotherapy does not preclude a strong serological response to the
MUC1 vaccine; however, the optimal time between completion of
chemotherapy and initiation of vaccine therapy is not known.

Fourth, we observed binding of IgM antibody to MCF-7 tumors cells
in seven of nine patients but minimal cell surface binding of IgG
antibody. Based on inhibition assays of sera from six patients, we
found that these antibodies bind primarily to the APDTRPA epitope of
the MUC1 peptide (53)
. Similar immunodominant epitopes
have been confirmed by others (24, 35)
. In our 30-amino
acid MUC1 immunogen, this epitope was located in both a medial and a
COOH-terminal position, yet the antibody bound primarily to the
COOH-terminal epitope. However in tumor cells, most of this epitope is
not expressed in the COOH-terminal position (54)
. This
explains our finding of modest cell surface reactivity in the face of
very high antibody titers against MUC1 peptide. It is probable that
other MUC1 peptides may generate antibodies against different epitopes.
For example, a clinical trial of a MUC1 peptide mannan conjugate
resulted in antibody production against other epitopes
(52)
. Therefore, further evaluation of different MUC1
peptides is warranted, with the goal of finding a synthetic peptide
that will more closely resemble the epitope exposure in patients.

The availability of immunological assays for precisely defining the
specificity and cell surface reactivity of vaccine induced serological
responses allows for analysis of a series of vaccines with relatively
small numbers of patients. Based on our results, we have designed
additional clinical trials aimed at improving the immunogenicity of the
current vaccine. First, we have recently completed a clinical trial to
assess a 32-amino acid MUC1 peptide containing two APDTRPA epitopes,
neither of which is at the terminal position. Second, an ongoing trial
will determine the immunogenicity of a 106-amino acid MUC1 peptide that
should naturally assume a β-helix configuration similar to the
proposed configuration on tumor cells. Third, another ongoing trial
will evaluate glycosylated MUC1 peptides because they may more closely
resemble the secondary structure of MUC1 on tumor cells.

Development of breast cancer vaccines involves determination of an
immunological response as well as clinical benefit. Therefore, the
results from these studies will allow for the selection of the optimal
MUC1 peptides for use in larger Phase II or III clinical trials.
Because of the heterogeneity of breast cancers, it is also possible
that an effective antitumor vaccine will require multiple antigenic
components, in addition to MUC1. These antigens are undergoing
evaluation in separate clinical trials. Although the optimal role of
vaccines for breast cancer therapy has not yet been identified, it is
reasonable to propose that clinical benefit be evaluated after adjuvant
chemotherapy, particularly in patients at high risk of recurrence, in
whom micrometastatic disease is likely.

Acknowledgments

We gratefully acknowledge the T cell analysis performed by Dr.
Olivera Finn and her colleagues at the University of Pittsburgh School
of Medicine. Lucy Dantis and the other nurses in the Immunology
Unit provided excellent patient care. We are also grateful to Jeannette
Chin for data management, Kristine Salerno for secretarial support, and
finally to the participants in the trial for their interest and
compliance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported in part by National Cancer Institute,
NIH, Grant PO1 CA33049 and the Milstein Family Foundation.

↵2 To whom requests for reprints should be
addressed, at Department of Medicine, Memorial Sloan-Kettering Cancer
Center, 1275 York Avenue, New York, NY 10021.